Novel polypeptides for isolating in vitro and preventing staphyloccocal infections on joint prostheses and other implanted foreign materials

ABSTRACT

The invention concerns novel polypeptides, or parts or variants of the novel polypeptides, the use of sequences encoding the polypeptides and the use of antibodies directed against the polypeptides in the field of in vitro diagnosis of a  Staphylococcus epidermidis  and/or  Staphylococcus aureus  infection on foreign material implanted in the body.

The present invention relates to a tool for serological multiparameterdiagnosis of staphylococcal infections on joint prostheses (for exampleinfection on a hip, elbow, knee, ankle prosthesis, etc.) and, moregenerally, diagnosis of staphylococcal infections on foreign materialimplanted in the body.

In particular, the invention comprises the identification of novelpolynucleotides and polypeptides as well as the production, optimisationand use thereof, on the one hand within the field of diagnosinginfections on foreign material (joint prostheses for example) involvingStaphylococcus aureus and/or Staphylococcus epidermidis and, on theother hand, within the field of producing vaccines against saiddifferent agents.

Infections on joint prostheses are a major problem in public health.Hundreds of thousands of joint prostheses are implanted worldwide everyyear and millions of people have one or more (Lew et Waldvogel, 1997,Osteomyelitis, N Engl J Med 336:999-1007). In the United States ofAmerica, it is estimated that approximately 430,000 total hip prostheses(THPs) and total knee prostheses (TKPs) are implanted every year(Berbari et al., 1998, Clin Infect. Dis., 27:1247-1254). In Norway andSweden, the only countries in which there is a register of prostheses,250,000 THPs were implanted within the space of ten years between 1987and 1996 (Lidgren, 2001, Joint prosthetic infections: a success story,Acta Othop Scand 72:553-556). In France, according to the French Societyof Orthopaedics, the number of prostheses implanted every year isapproximately 100,000 for THPs and 25,000 for TKPs (National Agency forAccreditation and Evaluation in Health, 2000). These figures willcontinue to rise in future owing to ageing populations (implantation ofTHPs) and obesity-related problems (implantation of TKPs) in developedcountries.

Despite the considerable progress made in recent years, prosthetic jointinfections (PJIs) are still a common complication affecting between 0.3and 1.8% of patients in the case of THPs (Berbari et al., 1998 [alreadycited]; Lidgren, 2001 [already cited]) and between 0.5 and 5% ofpatients in the case of TKPs (Johnson et Bannister, 1986, The outcome ofinfected arthroplasty of the knee, J Bone Joint Surg 68:289-291;Bengtson et Knutson, 1991, The infected knee arthroplasty, A 6-yearfollow-up of 357 cases, Acta Othop Scand 62:301-311; Eveillard et al,2002, Risque infectieux après implantation de prothèses de genou, Etudedes infections profondes pour une série continue de 210 prothèsestotales de genou en première intention, B E H No. 13). These rates areeven higher when it comes to replacement. Infection may occur at anytime after implantation of the prosthesis. In a large American study ofmore than 25,000 patients who have a THP or a TKP, the average period oftime between implanting the prostheses and diagnosis of infection was512 days, with periods ranging from 3 days to nearly 20 years and beingdistributed as follows: 20% of infections diagnosed within 3 monthsfollowing implantation, 40% between 3 months and 2 years, and 40% beyond2 years (Berbari et al., 1998 [already cited]).

PJI is a dangerous complication, resulting in the need for one or morerevision surgeries in combination with long-term antibiotherapy,however, long-term functional handicap, a risk of amputation or evendeath may also occur (Segawa et al, 1999, Infection after total kneearthroplasty, J Bone Joint Surg Am 81:1434-1445). The socio-economicimpact of a PJI is also extremely high, with an estimated cost of morethan 50,000 dollars per case (Sculco, 1995, The economic impact ofinfected joint arthroplasty, Orthopedics 18:871-873).

Staphylococci

Bacteria of the Staphylococcus genus are stationary, non-spore-forming,catalase-positive, oxidase-negative, gram-positive cocci groupedtogether in grape-like clusters. Observed by Pasteur in 1879 in furunclepus, staphylococci owe their name to Ogsten (1881) who isolated them inacute chronic abscesses.

S. aureus (more commonly known as golden staph) is generallydistinguished from other species of staphylococci. Unlike S. aureusthese other species do not produce any coagulase enzyme and, for thisreason, are grouped separately under the term “coagulase-negativestaphylococci” (CNSs).

Habitat of Staphylococci

The natural habitat of S. aureus is humans and warm-blooded animals. Inhumans, S. aureus preferably resides in nasal mucous; up to 30% ofadults permanently harbour S. aureus in their nostrils and 50% harbourit intermittently. Apart from the nose, S. aureus also resides on theskin and, in particular, in moist regions (armpits, perineum) and on thehands. Small amounts of S. aureus can also be found in the intestine.Lastly, the immediate environment of a human is also a source ofpotential contamination owing to the persistence of S. aureus in theexternal environment once it has been eliminated.

CNSs are the main commensals of skin together with coryne bacteria andpropionibacteria. The density of colonisation is greater in moistregions, such as the anterior portion of the nostrils, the perineum, thearmpits and the inguinal folds. Intra-human or inter-human transmissiongenerally occurs by direct contact (for example via the hands). Morerarely, transmission may be indirect from an environmental source(clothing, sheets, medical equipment).

Normally present in large quantities on skin and in mucosae, S.epidermidis and the other CNSs may contaminate superficial samples orsamples obtained by transcutaneous puncture, such as blood cultures, oreven deep perioperative samples taken during surgery. Consequently, CNSsare only considered responsible for an infection if this bacteria isfound multiple times in samples taken independently.

Role of Staphylococci in Human Pathology

S. aureus is responsible for many different infections. Cutaneo-mucosalinfections, such as folliculitis, impetigo, furuncles, anthrax, panaris,cellulitis, sinusitis or otitis are the most common. These infectionsmay be complicated by the loco-regional extension or hematogenousdiffusion of the bacteria. S. aureus thus causes septicaemia,endocarditis, pneumopothy, ostheomyelitis, arthritis and meningitis.These infections may be life-threatening, either per se (for example byattacking a heart valve) or in the case of associated toxic shock.

S. aureus has long been considered the only pathogenic staphylococcus inhumans, whilst CNSs were viewed as mere contaminants. The major role ofCNSs in human pathology has only recently been established, inparticular in patients with joint prostheses, artificial heart valves orimplantable devices such as vascular catheters or shunts for divertingcerebrospinal fluid.

S. epidermidis is the CNS which is most frequently isolated ininfections on foreign material. Other CNSs which are involved in thistype of infection include S. capitis, S. caprae, S. haemolyticus, S.lugdunensis, S. schleiferi, S. simulans and S. warneri.

Staphylococci are the main agents of PJIs and other infections onforeign materials.

Staphylococci are the bacteria most often found in PJIs and otherinfections on foreign materials, accounting for up to 75% of isolatedbacteria, S. aureus and S. epidermidis being the prevalent species (Lewet Waldvogel, 1997 [already cited]). According to studies, the group ofCNSs headed by S. epidermidis is alone responsible for 20 to 40% ofcases and ranges from being either the most common or second-most commonbehind S. aureus.

Apart from S. epidermidis, prevalent CNSs are S. capitis, S. caprae, S.haemolyticus, S. lugdunensis, S. schleiferi, S. simulans and S. warneri.Among these species, S. capitis, S. caprae and S. lugdunensis are themain CNSs, apart from S. epidermidis, responsible for prosthetic jointinfections and, more generally, osteoarticular infections on foreignmaterial (joint prostheses, osteosynthesis materials) (Rupp et Archer,1994, Coagulase-negative staphylococci: pathogens associated withmedical progress, Clin Infect Dis 19:231-243 ; Crichton et al, 1995,Subspecies discrimination of staphylococci from revision arthroplastiesby ribotyping. J Hosp Infect 30:139-147; Blanc et al, 1999, Infectionafter total hip replacement by Staphylococcus caprae. Case report andreview of the literature, Pathol Biol 47:409-413; Sampathkumar et al,2000, Prosthetic joint infection due to Staphylococcus lugdunensis. MayoClinic Proc 75:511-512; Weightman et al, 2000, Bone and prosthetic jointinfection with Staphylococcus lugdunensis, J Infect 40:98-99).

Generally, PJIs and other infections on foreign material caused by S.aureus are most often acute and suppurative owing to the numerousenzymes and toxins produced by this species (Nair et al, 2000, Advancesin our understanding of the bone and joint pathology caused byStaphylococcus aureus infection, Rheumatology (Oxford) 39:821-834),whilst CNS infections are mild and are often chronic (Von Eiff et al,2002, Pathogenesis of infections due to coagulase-negativestaphylococci, Lancet Infect Dis 2:677-685). However, this is not alwaysthe case: S. aureus infections may often be chronic whilst infectionscaused by CNSs, such as S. lugdunensis, may develop acutely(Sampathkumar et al, 2000 [already cited]; Weightman et al, 2000[already cited]).

Characteristics of Staphylococcal Infections on Foreign Material withBiofilm Formation

Staphylococcal infections on foreign material differ from conventionalinfections by the arrangement of bacteria in the form of biofilm(Costerton et al, 1999, Bacterial biofilms: a common cause of persistentinfections, Science 284:1318-22). This process has been described withreference to numerous staphylococci including S. aureus but has beenstudied, above all, in the case of S. epidermidis (Von Eiff et al, 2002[already cited]).

Biofilm is a complex three-dimensional structure which is connected tothe foreign material and in which the bacteria cells are embedded in apolysaccharide extracellular matrix called slime or glycocalyx (Daveyand O′Toole, 2000, Microbial biofilms: from ecology to moleculargenetics, Microbiology and Molecular Biology Reviews 64:847-867). Thisspecific structure may be formed by bacteria of the same species or ofdifferent species (Costerton et al, 1995, Microbial biofilms, Ann RevMed 49:711-745 ; Watnick and Kolter, 2000, Biofilms, city of microbes. JBacteriol 182:2675-2679). In comparison with their living consgeners infree (or ‘planctonic’) form, these bacteria are in a state of quiescenceindicated by a low level of metabolic activity (Yao et al, 2005,Genomewide analysis of gene expression in Staphylococcus epidermidisbiofilms: insights into the pathophysiology of S. epidermidis biofilmsand the role of phenol-soluble modulins in formation of biofilms, JInfect Dis 191:289-298).

Infections on foreign material associated with biofilm formation have anumber of features which distinguish them completely from conventionaltissue infections. These infections are most often paucibacillary(having few bacteria) and readily polymicrobial (combination of aplurality of species; for example, S. aureus and S. epidermidis or S.epidermidis and one or more other CNSs (Costerton et al, 1995 [alreadycited]; Watnick et Kolter, 2000 [already cited]). The bacteria have avery slow metabolism which keeps them in a state close to dormancy andthe genes which they express are different to those activated inplanctonic forms (Yao et al, 2005 [already cited]). The state ofdormancy of the bacteria and the presence of the biofilm significantlyreduce the inflammatory reaction and the attraction of immune cells atthe infection site. (Vuong et al, 2004, Crucial role forexopolysaccharide modification in bacterial biofilm formation, immuneevasion, and virulence, J. Biol. Chem, 279, 52:54881-54886; Fux et al,2003, Bacterial biofilms: a diagnostic and therapeutic challenge, ExpertRev Anti Infect Ther, 1:667-83). Lastly, for the same reasons, thebacteria are largely protected from the action of antibiotics (Costertonet al, 1995 [already cited]).

Current Methods for Diagnosing Staphylococcal Infections in a LaboratoryBacteriological Diagnosis

Nowadays, bacteriological diagnosis of a staphylococcal infection isalmost exclusively direct by isolating the bacteria in relevant samples.

Diagnosis is based on the following main steps:

(1) aseptic sampling (to reduce the risk of contamination by way of asimple commensal staphylococcus on the skin) carried out with noconcurrent antibiotherapy (after stopping or before starting antibiotictreatment).

(2) microscopic examination which enables common, gram-positive coccigrouped together in grape-like clusters to be observed. This examinationis completely insensitive and gives no indication of the speciesinvolved.

(3) culture on ordinary agar in the majority of cases or on a selectiveculture medium, CHAPMAN-type medium (which contains 7% NaCl, mannitoland a pH indicator) if the sample is potentially contaminated by otherbacteria.

(4) identification of the bacteria is based on the isolation of thefollowing features:

-   -   catalase (differentiation from streptococcus),    -   anaerobic glucose fermentation (differentiation from        micrococcus),    -   coagulase (differentiation from CNSs),    -   thermostable Dnase (which indicates the S. aureus species),    -   mini identification gallery (manual or automated)        (identification of CNS species)    -   optionally, molecular identification (for example, sequencing of        the sodA gene) (identification of CNS species).

(5) The diagnosis is completed by measuring sensitivity to antibiotics(antibiogram) given the frequency of resistance of staphylococci, inparticular in the case of hospital strains. The profile of sensitivityto antibiotics is also beneficial, although imperfect, for comparingdifferent strains (for true typing it is necessary to use moleculartechniques such as pulse field gel electrophoresis or multilocussequence typing).

The main drawback relates to the interpretation of the culture resultssince staphylococci are common contaminants. The general rule applied inorder to establish a diagnosis of infection is as follows:

S. aureus: at least one positive relevant sample (for example a bloodculture),

CNS: at least two independent relevant samples (for example two bloodcultures taken at two different times) which are positive for the samebacterium (i.e, in standard practice a bacterium of the same species andhaving the same sensitivity to antibiotics).

This threshold of two positive samples in the case of CNSs has recentlybeen called into question within the context of PJIs as it is consideredto be too nonspecific, and some authors maintain that, from now on, athreshold of three positive perioperative samples will be necessary todiagnose PJI caused by CNS (Atkins et al, 1998, Prospective evaluationof criteria for microbiological diagnosis of prosthetic-joint infectionat revision arthroplasty, J Clin Microbiol 36:2932-2939).

Serological Diagnosis

Nowadays, indirect diagnosis of staphylococcal infection by identifyingcirculating antibodies is largely undeveloped and is only relevant tothe diagnosis of some severe S. aureus infections outside foreignmaterial: endocarditis, septicaemia, haematogenous osteoarticularinfections (Söderquist et al, 1993, Staphylococcal a-toxin insepticaemic patients; detection in serum, antibody response andproduction in isolated strains, Serodiagn Immunother Infect Dis5:139-144; Nordin et al, 1995, Antibody response in patients withosteomyelitis of the mandible, Oral Surg Oral Med Oral Pathol OralRadiol Endod 79:429-435; Kanclerski et al, 1996, Serum antibody responseto Staphylococcus aureus enterotoxins and TSST-1 in patients withsepticaemia, J Med Microbiol 44:171-177; Colque-Navarro et al, 1998,Antibody response in Staphylococcus aureus septicemia—a prospectivestudy. J Med Microbiol 47:217-225; Colque-Navarro & Möllby, 1999,Usefulness of staphylococcal serology. J Med Microbiol 48:107-109; Elliset al, 2003, Role of staphylococcal enterotoxin A in a fatal case ofendocarditis, J Med Microbiol 52:109-112).

Current tests are based on the identification of antibodies directedagainst antigens specific to S. aureus or those produced by S. aureus,for example anti-α-toxin antibodies (or alpha antistaphylolysins),anti-β-ribitol teichoic acid, anti-enterotoxins and anti-staphylococcaltoxic shock syndrome toxin 1 (anti-TSST1), and anti-capsular antibodies.The antibodies are detected by haemolysis inhibition, byelectrosyneresis or by ELISA (Bornstein et al, 1992, Immune response tostaphylococcal toxins and ribitol teichoic acid in Staphylococcus aureusinfections, Med Microbiol Lett 1:111-119; Christensson et al, 1993,Diagnosing Staphylococcus aureus endocarditis by detecting antibodiesagainst S. aureus capsular polysaccharides types 5 and 8, J Infect Dis163:530-533; Kanclerski et al, 1996 [already cited]). These tests arelargely insensitive and nonspecific and give variable results dependingon the strains involved, in particular depending on the toxins whichthey produce (for example, greater response for positive enterotoxin Band/or C strains than for positive enterotoxin A and/or TSST1 strains)(Kanclerski et al, 1996 [already cited]). Lastly, there are no tests foridentifying the class of the antibodies produced, and in particularwhether they are IgG or IgA antibodies.

Both S. aureus and S. epidermidis or other CNSs are commensal bacteriacommonly found in humans (nasal membrane for S. aureus and skin for S.epidermidis and other CNSs) and identifying the antigens indicatinginfection is difficult. This difficulty has been confirmed by laboratorytests carried out by the applicant in which numerous staphylococcalantigens were not established as infection markers, the controlsresponding in the same proportions as the infected samples.

Diagnosis and Monitoring of Staphylococcal PJIs

It is vital to be able to diagnose a PJI with certainty. In fact,treatment of a PJI, which is involved and costly, is associated with asignificant impact on joint function and involves serious risks.Generally, treatment consists of extensive and complicated surgicaldebridement together with long-term tailored antibiotherapy. The mainsurgical options are replacing the prosthesis in a one-stage process ordebridement and retention of the prosthesis and re-implantation of theprosthesis in two stages, which requires longer periods ofhospitalisation (Garvin and Hanssen, 1995, Current concepts review.Infection after total hip arthroplasty. Past, present, and future, JBone Joint Surg 77-A:1576-1588). The mortality rate associated withsurgical procedure for PJI ranges from 0.4 to 1.2% for patients aged 65and from 2 to 7% for patients aged 80 (Lentino, Prosthetic jointinfections: bane of orthopedists, challenge for infectious diseasespecialists, Clin Infect Dis 36:1157-1161).

The risk of failure by way of infection is greater after revisionsurgery on PJIs, rising on average from 10 to 40% depending on location,the severity of lesions and the type of surgical treatment. Failure isnot always due to infection and, on the other hand, may involve anotherbacteria other than that which caused the first episode of PJI(associated bacteria not found in perioperative samples or bacteriainoculated accidentally during revision surgery).

A follow-up serological test makes it possible to give an earlydiagnosis of failure by identifying the nature of the infection and thebacteria involved.

In cases not involving acute or hyperacute PJIs, there are currently notests which make it possible to make a reliable pre-operative diagnosisof PJI and, in particular, to differentiate a PJI from asepticloosening, which is treated by simply exchanging the prosthesis in aone-stage process and which does not require any antibiotherapy. Theclinical signs are misleading: pain and inflammation are not specific toinfection and fever is usually absent. Simple radiography is insensitiveand the abnormalities indicating the presence of an infection take toolong to appear. Biological markers of inflammation, such as erythrocytesedimentation rate (ESR) or C-reactive protein (CRP), are insensitiveand nonspecific.

Nowadays, diagnosis of PJI can thus only be made with certainty byculturing perioperative samples or fluid aspirate, which involves asurgical procedure carried out under general anaesthetic (in some casesit is also possible to obtain true-cut-type samples without surgery butunder general anaesthetic).

This method poses several drawbacks:

it is not currently possible to give a pre-operative diagnosis of PJI oreven to establish which bacterium or bacteria are involved,

the methods for culturing perioperative samples are not standardised andrecent studies have shown that conventional methods lack sensitivity(Tunney et al, 1999. Detection of prosthetic hip infection at revisionarthroplasty by immunofluorescence microscopy and PCR amplification ofthe bacterial 16S rRNA gene, J Clin Microbiol 37:3281-3290),

the time required for culturing is particularly long in chronic cases,

interpretation of the culture results remains controversial: thethreshold of at least three independent positive samples suggested bythe OSIRIS group, Oxford provides excellent specificity (Atkins et al,1998 [already cited]), but may lack sensitivity, in particular whensamples are prepared in sub-optimal conditions.

None of the current diagnostic methods uses purified antigens toindicate a staphylococcal infection on a joint prosthesis and currentserological methods do not allow follow-up treatment orpost-implantation diagnosis to be carried out.

In this field there is thus an extreme need for a serological diagnosismethod in order to be able to carry out quick, cheap and non-invasivetests. Ideally, these tests will make it possible to identify thebacterium or bacteria involved and, in particular, will make it possibleto distinguish S. aureus, which has a significant ability to destroytissue, from other staphylococci.

Use of these tests may also make it possible to make an early diagnosisof PJI and thus enable better surgical management at an early stage ofinfection. This may be achieved by long-term serological monitoring,that is to say monitoring over a substantial period of time, of patientsafter prosthetic implantation (post-implantation diagnosis), inparticular in patients having a higher risk of PJI.

Lastly, in the future, if vaccinal approaches have been developed toprevent staphylococcal PJIs, in particular but not exclusively, thosecaused by S. aureus, tests of this type will be vital for detectingvaccination failures.

The object of the present invention is to overcome the drawbackspresented by the absence of serological tests enabling a staphylococcalinfection on foreign material, in particular on a joint prosthesis, tobe isolated by detecting circulating antibodies.

Another object of the present invention is to enable the detection ofcirculating antibodies which are directed against bacteria which arefound in a state of dormancy and are protected from the immune system bybeing present within a biofilm. A further object of the invention is toenable diagnosis of polymicrobial staphylococcal infections (that is tosay those involving a plurality of different species).

An additional object of the invention is to enable a more accurate andmore comprehensive analysis owing to the detection of different antibodyisotopes specific to polypeptides.

Definitions

The following definitions are given so as to facilitate theunderstanding of specific terms used within the description.

“Polynucleotide” means a polyribonucleotide or a polydeoxyribonucleotidewhich may be modified or unmodified DNA or RNA.

The term polynucleotide includes, in an non-limiting manner, singlestrand or double strand DNA, DNA formed of a mixture of one or moresingle strand regions and of one or more double strand regions, DNAwhich is a mixture of single strand regions, double strand regionsand/or triple strand regions, single strand or double strand RNA, RNAformed of a mixture of one or more single strand regions and of one ormore double strand regions, and hybrid molecules comprising DNA and RNAwhich may include single strand regions, double strand regions and/ortriple strand regions or a mixture of single strand and double strandregions. The term polynucleotide may also include RNA and/or DNAcomprising one or more triple strand regions. Strands in these regionsmay originate from the same molecule or from different molecules.Consequently, DNA or RNA with a backbone modified for reasons ofstability or otherwise are included in the term polynucleotides.Polynucleotide also means DNA and RNA containing one or more modifiedbases. Modified base means, for example, unusual bases such as inosine.The term polynucleotide also includes chemically, enzymatically ormetabolically modified polynucleotides. Polynucleotides also includeshort polynucleotides, such as oligonucleotides.

“Polypeptide” means a peptide, an oligopeptide, an oligomer or a proteincomprising at least two amino acids joined together by a normal ormodified peptide bond.

The term polypeptide includes short chains, known as peptides,oligopeptides and oligomers, and long chains known as proteins.

A polypeptide may be formed of amino acids other than the 20 amino acidscoded by human genes. A polypeptide may also be formed of amino acidsmodified by natural processes, such as by the post-translationalmaturation process or by chemical processes which are well known to theperson skilled in the art. The same type of modification may be presentat a plurality of locations on the polypeptide and anywhere within thepolypeptide: in the peptide backbone, in the amino acid chain or even atthe carboxy-terminal or amino-terminal ends.

A polypeptide may be branched following ubiquitination or cyclic with orwithout branching. These types of modification may be the result of anatural or synthetic post-translational process, these processes beingwell known to the person skilled in the art.

Modification of a polypeptide means, for example, acetylation,acylation, ADP-ribosylation, amidation, covalent binding of flavin,covalent binding of a heme, covalent binding of a nucleotide or of anucleotide derivative, covalent binding of a lipid or of a lipidderivative, covalent binding of a phosphatidylinositol, covalent ornon-covalent cross linking, cyclisation, formation of a disulphide bond,demethylation, the formation of cysteine, the formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, theformation of a GPI anchor, hydroxylation, iodisation, methylation,myristoylation, oxidation, the proteolytic process, phosphorylation,prenylation, racemisation, seneloylation, sulphation, amino acidaddition such as arginylation or ubiquitination (PROTEINS STRUCTURE ANDMOLECULAR PROPERTIES, 2^(nd) Ed., T. E. Creighton, W.H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs 1-12 in POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) andRattan et al., Protein Synthesis: Posttranslational Modifications andAging, Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

“Percentage identity” between two polynucleotide or polypeptidesequences means the percentage of identical nucleotides or amino acidsin the two sequences to be compared and is obtained after achieving thebest alignment possible, this percentage being purely statistical andthe differences between the two sequences being randomly distributedover their entire length. Comparisons between two polynucleotide orpolypeptide sequences are conventionally carried out by comparing thesesequences after having optimally aligned them, said comparison beingcarried out per segment or per “comparison window” in order to identifyand compare the local regions with sequence similarity. This comparisonmay be carried out by means of a program, for example the EMBOSS-Needleprogram (Needleman-Wunsch global alignment) using the BLOSUM62matrix/Gap opening penalty 10.0 and Gap extension penalty 0.5 (Needlemanet Wunsch (1970), J. Mol. Biol. 48, 443-453 and Kruskal, J. B. (1983),An overview of sequence comparison, In D. Sankoff and J. B. Kruskal,(ed), Time warps, string edits and macromolecules: the theory andpractice of sequence comparison, pp. 1-44 Addison Wesley).

The percentage identity is calculated by determining the number ofidentical positions for which the nucleotide or the amino acid isidentical between the two sequences, by dividing this number ofidentical positions by the total number of positions within thecomparison window and by multiplying the result by 100.

A polynucleotide having, for example, an identity of at least 95% withthe polynucleotide of SEQ ID No. 1 is thus a polynucleotide comprising,at most, 5 modified nucleotides out of 100 nucleotides compared withsaid sequence. In other words, up to 5% of the nucleotides in thesequence of SEQ ID No. 1 can be deleted or substituted by anothernucleotide, or up to 5% of the total number of nucleotides in thesequence of SEQ ID No. 1 may be inserted into said sequence. Thesemodifications may be located at the 3′ and/or 5′ ends, or anywherebetween these ends, at one or more locations.

Similarly, a polypeptide having an identity of at least 95% with thepolypeptide of SEQ ID No. 2 is a polypeptide comprising, at most, 5modified amino acids out of 100 amino acids compared with said sequence.In other words, up to 5% of the amino acids in the sequence of SEQ IDNo. 2 can be deleted or substituted by another amino acid or up to 5% ofthe total number of amino acids in the sequence of SEQ ID No. 2 may beinserted into said sequence. These changes to the sequence may belocated at the amino-terminal and/or carboxy-terminal positions of theamino acid sequence or anywhere between these terminal positions, at oneor more locations. (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987).

With regard to the term “similarity”, this is calculated in the same wayas identity except that amino acids which are not identical but whichhave common physico-chemical characteristics are considered to beidentical.

“Host cell” means a cell which has been transformed or transfected, oris capable of being transformed or transfected, by an exogenouspolynucleotide sequence.

“Culture medium” means the medium in which the polypeptide of theinvention is purified. This medium may be formed by the extracellularmedium and/or the cellular lysate. Methods which are well known to theperson skilled in the art also make it possible restore the activeconformation of the polypeptide if the conformation of said polypeptidewas modified during isolation or purification.

“Function” means the biological activity of a polypeptide or of apolynucleotide.

The function of a polypeptide in accordance with the invention is thatof a Staphylococcus epidermidis and/or Staphylococcus aureus and/orStaphylococcus caprae antigen, and the function of a polynucleotide inaccordance with the invention is that of coding said polypeptide. Thefunction of a combination of antigens is to make it possible to diagnosean infection on foreign material and, in particular, on anosteoarticular prosthesis, but also to carry out post-implantationfollow-up checks, therapeutic follow-up checks and, lastly, to recognisewhether an active or acute infection is involved, for example bydetecting different antibody isotopes.

“Antigen” means any compound which, either alone or in combination withan adjuvant or carrier, is capable of inducing a specific immuneresponse. This definition also includes any compound exhibitingstructural similarity with said antigen capable of inducing animmunological response directed against said antigen.

“Structural similarity” means a similarity of both the primary structure(sequence) and of the secondary structure (structural elements), of thetertiary structure (three-dimensional structure) or of the quaternarystructure (association of a plurality of polypeptides in a singlecomplex) (BIOCHEMISTRY, 4^(th) Ed, L. Stryer, New York, 1995).

A “variant” of what is known as an initial polynucleotide or of what isknown as an initial polypeptide means, respectively, a polynucleotide ora polypeptide which differs therefrom by at least one nucleotide or oneamino acid, but which maintains the same intrinsic properties, that isto say the same function.

A difference in the polynucleotide sequence of the variant may or maynot alter the amino acid sequence of the polypeptide which it codescompared with an initial polypeptide. However, by definition, thesevariants must confer the same function as the initial polynucleotidesequence, for example code a polypeptide having an antigenic function.

The variant polynucleotide or variant polypeptide generally differs fromthe initial polynucleotide or initial polypeptide by one (or more)substitutions, additions, deletions, fusions or truncations or by acombination of a plurality of these modifications. An unnatural variantof an initial polynucleotide or of an initial polypeptide may beobtained, for example, by site-directed mutagenesis or by directsynthesis.

A “polynucleotide sequence complementary to the polynucleotide sequence”is defined as a polynucleotide which may be hybridised with saidpolynucleotide sequence under stringent conditions.

Generally, but not necessarily, “stringent conditions” means chemicalconditions enabling hybridisation when the polynucleotide sequences havean identity of at least 80%.

These conditions may be obtained in accordance with methods which arewell known to the person skilled in the art.

“Antibodies” means humanised, single-chain, chimeric monoclonal andpolyclonal antibodies as well as Fab fragments, including products ofFab or immunoglobin expression library. It is also possible to use otherimmunospecific molecules in place of antibodies, for example T-cellreceptors (TCRs) as recently described (Li et al, 2005, Directedevolution of human T-cell receptors with picomolar affinities by phagedisplay, Nat Biotechnol, 23:349-54) or molecules selected for theirspecific binding ability, for example by directed evolution (see forexample Conrad and Scheller. 2005. Considerations on antibody-phagedisplay methodology, Comb Chem High Throughput Screen, 8:117-26).

An immunospecific antibody may be obtained by administering a givenpolypeptide to an animal followed by recovery of the antibodies producedby said animal by way of extraction from its bodily fluids. A variant ofsaid polypeptide, or host cells expressing said polypeptide may also beadministered to the animal.

The term “immunospecific” applied to the term antibody, in relation to agiven polypeptide, means that the antibody has a greater affinity forthis polypeptide than for other polypeptides known from the prior art.

“Positive” serum means a serum containing antibodies produced followingan S. epidermidis or S. aureus infection on a joint prosthesis andidentified by way of their binding to the polypeptides (antigens) of theinvention.

“Sensitivity” means the proportion of infected patients diagnosed inaccordance with the prior art and said to be positive by way of thediagnostic procedure according to the invention.

“Specificity” means the proportion of blood donors, tested as controls,who underwent the diagnostic procedure in accordance with the inventionand who were said to be negative by way of the diagnostic procedureaccording to the invention.

The present invention achieves the objects detailed above by providingnovel polynucleotides and novel polypeptides as well as fragments whichhave proven to be more relevant and/or more sensitive and/or morespecific than the entire protein.

The invention relates to the in vitro use of at least one of theproteins of the sequence of SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34 or 36, of part or variants of saidprotein, of host cells comprising vectors including a polynucleotidecoding at least one of said proteins or a variant of said proteins, inthe production of antibodies and within the field of in vitro diagnosisof Staphylococcus epidermidis and/or S. aureus. The invention alsorelates to a diagnostic kit and a pharmaceutical composition.

Use of Polypeptides

The applicant's laboratory is aware of polypeptides produced by S.epidermidis or S. aureus which do not allow prognosis of an infection onforeign material, in particular on a joint prosthesis (for example Sepidermidis recombinant protein “Thimet oligopeptidase-like protein”(Q8CPS6).

However, the applicant unexpectedly found that a prognosis of this typeis in fact possible by using other polypeptides produced by S.epidermidis or S. aureus specifically identified by the applicant, andeven by using polypeptide fragments produced by S. epidermidis or S.aureus which, when complete, do not allow this prognosis.

The identification of the polypeptides according to the invention is theresult of close examination and in-depth studies and was not possibleusing the sequences produced by the genome research programmes into S.epidermidis or S. aureus.

The present invention thus relates to the use, in the production ofantibodies and within the field of in vitro diagnosis of Staphylococcusepidermidis and/or S. aureus infections, of at least one polypeptidecomprising:

amino acid sequence of SEQ ID No. 2 (known as protein E4), coded bypolynucleotide sequence of SEQ ID No. 1; or

-   -   amino acid sequence of SEQ ID No. 4 (known as protein 2B6),        coded by polynucleotide sequence of SEQ ID No. 3; or    -   amino acid sequence of SEQ ID No. 6 (known as protein F2), coded        by polynucleotide sequence of SEQ ID No. 5; or    -   amino acid sequence of SEQ ID No. 8 (known as protein 3F7),        coded by polynucleotide sequence of SEQ ID No. 7; or    -   amino acid sequence of SEQ ID No. 10 (known as protein 2D6B1),        coded by polynucleotide sequence of SEQ ID No. 9; or    -   amino acid sequence of SEQ ID No. 12 (known as protein JR7),        coded by polynucleotide sequence of SEQ ID No. 11; or    -   amino acid sequence of SEQ ID No. 14 (known as protein JR12),        coded by polynucleotide sequence of SEQ ID No. 13; or    -   amino acid sequence of SEQ ID No. 16 (known as protein JR5),        coded by polynucleotide sequence of SEQ ID No. 15; or    -   amino acid sequence of SEQ ID No. 18 (known as protein 3A7),        coded by polynucleotide sequence of SEQ ID No. 17; or    -   amino acid sequence of SEQ ID No. 20 (known as protein 3B6),        coded by polynucleotide sequence of SEQ ID No. 19; or    -   amino acid sequence of SEQ ID No. 22 (known as protein 3D5),        coded by polynucleotide sequence of SEQ ID No. 21; or    -   amino acid sequence of SEQ ID No. 24 (known as protein 3E5),        coded by polynucleotide sequence of SEQ ID No. 23; or    -   amino acid sequence of SEQ ID No. 26 (known as protein 3F3),        coded by polynucleotide sequence of SEQ ID No. 25; or    -   amino acid sequence of SEQ ID No. 28 (known as protein 3G3),        coded by polynucleotide sequence of SEQ ID No. 27; or    -   amino acid sequence of SEQ ID No. 30 (known as protein 3H3),        coded by polynucleotide sequence of SEQ ID No. 29; or    -   amino acid sequence of SEQ ID No. 32 (known as protein 3H4),        coded by polynucleotide sequence of SEQ ID No. 31; or    -   amino acid sequence of SEQ ID No. 34 (known as protein 3D7),        coded by polynucleotide sequence of SEQ ID No. 33; or    -   amino acid sequence of SEQ ID No. 36 (known as protein 3C7),        coded by polynucleotide sequence of SEQ ID No. 35.

The present invention also relates to the use of at least onepolypeptide comprising:

a) part of the amino acid sequence of SEQ ID No. 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 having the same function assaid sequence, or

b) an amino acid sequence having at least 60% identity, preferably atleast 80% identity and most preferably at least 90% identity with one ofthe amino acid sequences of SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34 or 36 or with part of the sequencedefined under a), and having the same function as said sequence.

The polypeptides in accordance with the invention may thus comprisevariants of amino acid sequences of SEQ ID No. 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36.

The polypeptide sequence of SEQ ID No. 4, known as protein 2B6, coded bythe sequence of SEQ ID No. 3, comprises amino acids 1 to 184 of thesequence of SEQ ID No. 2 and has the same function as the sequence ofSEQ ID No. 2.

The polypeptide sequence of SEQ ID No. 6 (known as protein F2), coded bythe polynucleotide sequence of SEQ ID No. 5, has 60% identity and 78%similarity with the sequence of SEQ ID No. 2.

Other variants of the sequences of SEQ ID Nos. 2, 4, and 6 are, forexample, part of the AtlC protein (3F7 (SEQ ID No. 8) for example) codedby the bacterium Staphylococcus caprae.

Owing to the polypeptides defined above, the invention enables detectionof antibodies which are produced naturally during staphylococcalinfections on joint prostheses.

The invention further relates to the use of polypeptides according tothe invention to periodically detect, in vitro, antibodies directedagainst S. epidermidis and/or S. aureus and thus to monitor theprogression of the pathology and the effect of treatment given to apatient.

The biological samples tested may be samples of blood, urine, saliva,fluid obtained via serological puncture (for example cerebrospinalfluid, pleural fluid or joint fluid) or of one of their constituents(for example serum).

A Staphylococcus epidermidis and/or S. aureus infection is diagnosed invitro, for example using conventional tests for testing immunologicalreactions, such as ELISA or Western Blot tests. These tests use one ofthe polypeptides according to the invention which bonds, specifically,to any serum antibodies directed against Staphylococcus epidermidisand/or S. aureus present in the biological samples.

Use of Expression Vectors and Host Cells

The present invention also relates to the use of a polypeptide preparedby culturing a host cell comprising a recombinant vector having,inserted, a polynucleotide coding said polypeptide.

Numerous expression systems may be used, such as chromosomes, episomesand derived viruses. More particularly, the recombinant vectors used maybe derived from bacterial plasmids, transposons, yeast episomes,insertion elements, chromosomal elements of yeasts, viruses such asbaculoviruses, papillonna viruses such as SV40, vaccinia viruses,adenovirusus, fox pox viruses, pseudorabies viruses, and retroviruses.

These recombinant vectors may also be derivatives of cosmids orphagemids. The polynucleotide sequence may be inserted into therecombinant expression vector by methods well known to the personskilled in the art.

The recombinant vector may comprise polynucleotide sequences formonitoring the regulation of the polynucleotide expression as well aspolynucleotide sequences enabling expression and transcription of apolynucleotide according to the invention and translation of apolypeptide according to the invention, these sequences being selectedas a function of the host cells used.

The introduction of the recombinant vector into a host cell may becarried out in accordance with methods which are well known to theperson skilled in the art, such as transfection by calcium phosphate,transfection by cationic lipids, electroporation, transduction orinfection.

The host cells may be, for example, bacterial cells, such asstreptococcal cells, staphylococcal cells, Escherichia coli or Bacillussubtilis cells; fungus cells, such as yeast cells and Aspergillus cells;Streptomyces cells; insect cells, such as Drosophilia S2 and spodopteraSf9 cells; animal cells, such as CHO, COS, HeLa, C127, BHK, HEK 293cells or even vegetable cells.

The polypeptide may be purified from host cells in accordance withmethods which are well known to the person skilled in the art, such asprecipitation using chaotropic agents, such as salts, in particularammonium sulphate, ethanol, acetone or tricholoroacetic acid, or bymeans such as acid extraction, ion exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography or exclusionchromatography.

Use of Polynucleotides

The present invention also relates to the use, in the production ofantibodies and in the diagnosis of a Staphylococcus epidermidis and/oraureus infection, of at least one polynucleotide comprising thepolynucleotide sequence of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33 or 35, coding, respectively, protein 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36.

The invention also relates to the use of at least one polynucleotidecomprising:

a) part of the sequence of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33 or 35, and having the same function as saidsequence, or

b) a polynucleotide sequence having at least 60% identity, preferably atleast 80% identity, and most preferably at least 90% identity with thepolynucleotide sequence of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33 or 35 or with the sequence part as definedunder a), and having the same function as said sequence, or

c) a polynucleotide sequence complementary to the polynucleotidesequence of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33 or 35 or to the sequence part defined under a) or to thesequence defined under b).

The polynucleotide sequence of SEQ ID No. 3 comprises nucleotides 1 to552 of the sequence of SEQ ID No. 1 and has the same function as saidsequence.

The polynucleotide sequence of SEQ ID No. 5 (known as F2 and extractedfrom the Staphylococcus aureus genome) is a variant of the sequence ofSEQ ID No. 1.

Other variants are, for example, polynucleotide sequences codingproteins of the autolysin family (for example the sequence of SEQ ID No.7, a variant of the sequence of SEQ ID No. 1 coding part of the AtlCprotein, known as 3F7 and present in the Staphylococcus capraebacterium). The polynucleotides according to the invention may thuscomprise variants of one of the polynucleotide sequences of SEQ ID No.1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35.

The polynucleotides of the invention may be obtained by standard DNA orRNA synthesis methods.

The polynucleotides according to the invention may also comprisepolynucleotide sequences, such as the non-coding 5′ and/or 3′ sequences,for example transcript sequences, untranslated sequences, splicingsignal sequences, polyadenylated sequences, ribosome-binding sequencesor even RNAm stabilising sequences.

Use of the Antibodies According to the Invention

The invention also relates to the use of antibodies, according to theinvention, for in vitro detection in biological samples of the presenceof S. epidermidis and/or S. aureus antigens.

The invention further relates to the use of antibodies according to theinvention to periodically detect, in vitro, S. epidermidis and/or S.aureus antigens and thus to monitor the progression of the pathology andthe effect of treatment given to a patient.

Immunospecific antibodies may be obtained by administering a polypeptideaccording to the invention, one of its fragments, an analogue or anepitopic fragment, or a cell expressing said polypeptide to a preferablynon-human mammal, in accordance with methods well known to the personskilled in the art.

In order to prepare monoclonal antibodies, conventional methods forproducing antibodies from cellular lines, such as the hybridomal method,the trioma method, the human B cell hybridomal method and the EBVhybridomal method may be used.

Antibody Isotopes and Affinity

Immunoglobulins (or antibodies) are formed of two different polypeptidechains: two light chains (L) of [kappa] or [lambda] isotopes, and twoheavy chains (H) of [gamma], [alpha], [mu], [delta] or [epsilon]isotopes. These four chains are covalently bonded by disulphide bonds.The two chain types H or L have regions which contribute to the bindingof antigens and may vary greatly from one immunoglobulin to another.These regions determine the affinity of the antibodies for their ligand.

Heavy chain isotopes make it possible to define the class of theimmunoglobulin. There are thus five antibody isotopes (IgA, IgM, IgD,IgE and IgG). Sub-classes, for example IgG1, IgG2, IgG3 and IgG4 aredefined by the differences in the heavy chain sequence. The differentclasses of immunoglobulins have their own physico-chemical propertiesand their synthesis depends directly on the phases and activation levelsof the immune response.

In humans, the IgGs are the main class of immunoglobulins: their serumconcentration in adults varies from 8 to 16 g/l. Their plasma half-lifeis approximately three weeks.

IgAs are the second most common class of serum immunoglobulins afterIgGs in terms of concentration (2 to 4 g/l). In contrast, they are thepredominant class of immunoglobulins in secretions (respiratory,salivary, digestive secretions, etc, milk, colostrum, tears). Withregard to structure, IgAs are distinct by existing in a plurality ofmolecular forms:

in serum, IgAs may be present in the form of monomers (predominant form)or in the form of dimers associated with a J chain (junction chain). TheJ chain is a cysteine-rich peptide of 137 amino acids (molecular weight:15,000 Da), of plasmocyte origin; sub-class IgA1 is predominant inserum.

in secretions, IgAs, known as secretory IgAs, are in the form of dimers:they are thus associated with a J chain but they also comprise asecretory component. IgAs produced by B lymphocytes are captured byepithelial cells by a receptor (polyIgR) arranged at the basal pole ofthe cell. It is during the transfer of IgA through the epithelial celltowards the apical pole of the cell that the secretory component(molecular weight: 70,000 Da) or secretory piece is added. The role ofthe secretory piece is to protect the secretory IgAs from proteolyticenzymes present in the secretions.

Similarly to IgAs, IgMs may exist in two distinct molecular forms:

a monomeric form: this is the form in which IgMs are synthesised andinserted into the membrane of B lymphocytes;

a pentameric form: this is the form in which IgMs are secreted. The fivebase monomers are connected by disulphide bonds. Furthermore, a J chainconnects the ends of two monomers.

IgDs account for less than 1% of serum immunoglobulins. They are usuallycoexpressed with IgMs at the surface of B lymphocytes where they appearto play the role of antigen receptors.

In a normal subject IgEs are only present in trace form.

Kinetics of Appearance of Antibodies and Affinity

The kinetics of appearance of specific antibodies and of a given isotopeis still not understood well. Generally, B cells known as naive B cellssynthesise different IgMs which constitute a large source of moleculesable to bind antigens (Steven A. Frank, Immunology and Evolution ofInfectious Disease, (2002), Princeton University Press, Princeton, USA).When first exposed to an antigen, said antigen thus binds with a weakaffinity to different IgMs of the immune system. However, thisinteraction stimulates division of the corresponding naive B cell. Therapidity of division increases with the strength of the affinity of theIgM which enables an initial selection of the best clones. Furthermore,during division genetic rearrangements allow the affinity of theantibodies to increase. The constant region of the immunoglobulins alsochanges so as to produce IgGs in the circulatory system and IgAs atmucous surfaces (Steven et Frank 2002, Immunology and Evolution ofInfectious Disease, Princeton University Press, Princeton, USA).

The person skilled in the art may thus deduce that the presence of oneor more antibody classes and the specific affinity of the antibodieshave different implications from a medical diagnostic point of view whendifferentiating, in the case of recurrent or non-recurrent infections,between an active, acute, chronic, latent and recent infection.

Kits

The invention also relates to in vitro diagnostic kits comprising atleast one of the polypeptides according to the invention, or at leastone of the polynucleotides coding said polypeptides, as well as in vitrodiagnostic kits comprising at least one of the antibodies according tothe invention.

Vaccines

The present invention also relates to a pharmaceutical composition whichcan be used as a vaccine and contains, as an active ingredient, at leastone polypeptide according to the invention or a polynucleotide or arecombinant vector or a host cell according to the invention.

EXPERIMENTAL PART A) Protocols for Producing Antigens A.1. Cloning ofthe Sequence Coding Polypeptides of SEQ ID No. 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36.

Genes coding polypeptide sequences, which are antigens, are obtained byPCR amplification from the genomic DNA of Staphylococcus epidermidis(WHO 12 strain, ATCC 12228), Staphylococcus caprae (ATCC 35538) orStaphylococcus aureus (MU50 strain, ATCC 700699 or MW2) bacteria byusing, respectively, specific primers of the sequences of SEQ ID No. 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35.Specific primers means short nucleotide sequences capable ofhybridisation in a specific manner, owing to the base-pairing rule, onthe DNA strand or on its complementary strand, these primers beingselected by the person skilled in the art so as to include the DNAsequence and to amplify it specifically (for example SEQ ID No. 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35).

The corresponding fragment thus amplified is cloned into a vector inaccordance with conventional methods well known to the person skilled inthe art. This vector allows the production of cloned proteins under thecontrol of an isopropyl thiogalactoside (IPTG) inducible promoter. Thecloned proteins correspond to the polypeptides of SEQ ID No. 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36.

A.2. Expression of Proteins

An Escherichia coli strain is transformed by the aforementionedexpression vectors. The selected bacteria are cultured overnight at 30°C., with stirring, in 30 ml of Luria Bertani medium (LB, J. Miller, “Ashort Course in Bacterial Genetics”, Cold Spring Harbor LaboratoryPress, 1992) containing ampicillin at a final concentration of 100μg/ml. The next day, the culture is diluted at a ratio of 1:50 in afinal volume of 1 litre of LB medium supplemented with ampicillin at afinal concentration of 100 μg/ml and incubated at 30° C. with stirring.When the turbidity of the culture reaches an absorbance value at 600 nm(A600) of approximately 0.7, the production of the protein is induced byisopropyl thiogalactoside (IPTG) at a final concentration of 0.1 mM. Thebacteria are harvested by centrifugation (10 minutes at 1400×g and at 4°C.) when the turbidity of the culture reaches an A600 of approximately1.5.

A.3. Purification of Proteins

After centrifugation, the cells are resuspended in a 20 mM Tris-HClbuffer at pH 8.0 containing sucrose at 0.5 mM, then treated withlysozyme (0.2 g/l) in the presence of 12.5 mM ofethylenediaminetetraacetic acid (EDTA), DNase, RNase and PMSF. Thesuspension is incubated for 30 minutes at 4° C. and then centrifuged for10 minutes at 4° C. at 15,500×g. The pellet is frozen at −20° C. for atleast one night.

A.4. Example: Purification of 3C7 (SEQ ID No. 36)

After thawing, the bacteria are placed in a Mes 25 mM buffer at pH 6.0,then sonicated for 20 seconds in ice, four times. After centrifugationat 15,500×g at 4° C. for 30 minutes, the supernatant is filtered on amembrane with a porosity of 0.22 μm. The filtrate is then deposited on acation-exchange column (for example 12 ml SP-Sepharose, AmershamBiosciences). After washing the column, the protein is eluted with alinear gradient of 0 to 1 M of NaCl in Mes 25 mM buffer at pH 6.0 in 20column volumes. The fractions containing the protein are combined andthe proteins are precipitated by ammonium sulphate at a finalconcentration of 0.6 g/l. The solution is left for at least one night at4° C. and then centrifuged for 30 minutes at 20,800×g. The pellet isthen taken up in the smallest volume possible (generally 300 μl of 50 mMNa₂HPO₄/NaH₂PO₄ buffer at pH 8.0 containing 100 mM NaCl and thendeposited on a gel filtration column, for example Superdex HR75-10/30,Amersham). The eluted fractions containing the protein are combined andglycerol is added until a final concentration of 20% is obtained. Thepurified proteins are then stored at −20° C. until they are used in thetests.

The concentrations of the proteins are determined spectrophotometricallyfrom the absorption coefficients calculated using the Pace method (Paceet al, 1995, How to measure and predict the molar absorption coefficientof a protein, Protein Science 4, 2411-2423). The purity of the proteinsis checked by SDS-PAGE electrophoresis analysis and by massspectrometry.

B) In vitro Diagnostic Test

Sera obtained from patients having had a documented osteoarticularinfection (laboratory collection) caused by:

Staphylococcus epidermidis (at least three positive perioperativesamples);

Staphylococcus aureus (at least one positive perioperative sample);

bacteria other than staphylococci (at least three positive perioperativesamples) were used.

The control sera were sera obtained from blood donors (laboratorycollection).

Example B1 Protocol of the Test for Polypeptides of SEQ ID Nos 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34 (ELISA)

The binding of the antibodies present in the sera was assessed usingELISA tests. The ELISA plates were left overnight at 4° C. in thepresence of 0.5 μg of purified antigen (recombinant protein E4) in aphosphate buffered saline (PBS). After four washes with PBS containing0.05% polyoxyethylene sorbitan (Tween), the plates were saturated forone hour at 37° C. in PBS-Tween containing 5% semi-skimmed milk (250 μlper well). Four new washes were carried out and then 100 μl of eachpositive serum, at the appropriate dilution ratio (i.e. 1:3000 for E4)in PBS-Tween buffer containing 5% semi-skimmed milk, were added to eachwell. The plate was then left at 25° C. for 30 minutes. After four newwashes, goat anti-human immunoglobulin G, M, or A (secondary) antibodiesor, simultaneoulsy, goat anti-human immunoglobulin G and/or A and/or M(secondary) antibodies labelled with alkaline phosphatase (for example170-6462, Biorad) were added for 30 minutes at 25° C. after having beendiluted in accordance with the supplier's protocol in PBS-Tween buffercontaining 5% semi-skimmed milk. Four new washes were carried out andthen 100 μl of pNPP (p-nitrophenyl phosphate) substrate, for exampleA-3469, Sigma were added. The absorbance at 405 nm of each of the wellswas measured after incubation for 30 minutes at 37° C.

Results and Interpretation

The tests were carried out on recombinant proteins obtained fromindependent purifications.

Typical results are shown in Table 1 (results according to the inventionfor polypeptides E4, F2 and 2D6-B1 with secondary antibodies recognisingthe total immunoglobulins (Ig G, A, and M) present in the patient sera).

TABLE 1 results (ELISA) obtained using anti-IgGAM antibodies assecondary antibodies Polypeptide tested E4 F2 2D6B1 Percentage of“positive” sera from the 47.0% 50.0% 40.0% 15 sera of patients infectedwith S. epidermidis diagnosed according to the prior art and whounderwent the diagnostic procedure according to the present inventionPercentage of “positive” sera from the 26.0% 25.0% 25.0% 16 sera ofpatients infected with S. aureus diagnosed according to the prior artand who underwent the diagnostic procedure according to the presentinvention Percentage of “negative” sera from the 95.0% 98.0% 96.8% 96blood donor sera tested as controls

It can be seen from Table 1 that the polypeptides of the invention maybe used to isolate staphylococcal infections on joint prostheses.

Example B2 Results Obtained by Detecting of Antibody Isotypes in Sera(ELISA)

The use of some polypeptides, for example 2D6-B1 (Table 2) to detectimmunoglobulins of a specific isotype is of particular relevance.

TABLE 2 Examples of results (ELISA) using anti-IgA antibodies assecondary antibodies Polypeptide tested 2D6B1 Percentage of “positive”sera from the 15 sera of patients 47.0% infected with S. epidermidisdiagnosed according to the prior art and who underwent the diagnosticprocedure according to the present invention Percentage of “positive”sera from the 16 sera of patients 31.0% infected with S. aureusdiagnosed according to the prior art and who underwent the diagnosticprocedure according to the present invention Percentage of “negative”sera from the 96 blood donor sera 97.0% tested as controls

It follows that the polypeptides may be used to indicate the presence ofsome antibody isotypes in the case of staphylococcal infections on jointprostheses.

Example B3 Combination of Polypeptides for in vitro Diagnosis of PJI

The combined use of a plurality of antigens and/or the detection of aplurality of antibody isotypes makes it possible to increase thesensitivity of the method (Tables 3 and 4).

Polypeptide 2B6 is very specific to S. epidermidis infections. Incontrast with 2B6, polypeptide 2D6-B1 is not specific to a particularspecies and “positive” results may be observed with sera from patientshaving a PJI caused by other species of Staphylococcus (for example S.lugdunensis). This polypeptide is therefore “specific” to thestaphylococcus genus and not to a specific species. It is thus possible,by using different polypeptides (or by detecting different antibodyisotypes), to differentiate between species.

TABLE 3 Results (ELISA) obtained using combinations of differentpolypeptides according to the invention to detect immunoglobulins IgG, Aor M Polypeptide tested 2B6 2D6B1 2B6 or 2D6B1 Percentage of “positive”sera from 67.0% 40.0% 87.0% the 15 sera of patients infected with S.epidermidis diagnosed according to the prior art and who underwent thediagnostic procedure according to the present invention Percentage of“positive” sera from 11.0% 25.0% 37.5% the 16 sera of patients infectedwith S. aureus diagnosed according to the prior art and who underwentthe diagnostic procedure according to the present invention Percentageof “negative” sera from 99.0% 96.8% 96.0% the 96 blood donor sera testedas controls

TABLE 4 Results (ELISA) obtained by using 2D6B1 as a polypeptide and bydetecting antibodies using different secondary antibodies Secondaryantibodies used IgA IgG IgG or A Percentage of “positive” sera from47.0% 40.0% 67.0% the 15 sera of patients infected with S. epidermidisdiagnosed according to the prior art and who underwent the diagnosticprocedure according to the present invention Percentage of “positive”sera from 31.0% 25.0% 37.0% the 16 sera of patients infected with S.aureus diagnosed according to the prior art and who underwent thediagnostic procedure according to the present invention Percentage of“negative” sera from 97.0% 96.0% 96.0% the 96 blood donor sera tested ascontrols

Example B4 Identification of PJIs Not Diagnosed by CulturingPerioperative Samples

These tests were carried out using sera from patients who underwentsurgery for a suspected staphylococcal infection on a joint prosthesis,but who were considered, from a bacteriological point of view, to beuninfected in accordance with results following culture of deepperioperative samples obtained during revision surgery on the prosthesis(only one or two perioperative samples positive for CNS).

Table 5 shows an example of typical results obtained by detecting thetotal anti-2D6B1 immunoglobulins in 7 patients having had one or twoperioperative samples which were positive for S. epidermidis.(laboratory collection). The control sera were sera obtained from blooddonors (laboratory collection).

TABLE 5 Type of prosthesis ELISA value Patient 1 TKP 2.026 Patient 2 THP0.151 Patient 3 TKP 0.302 Patient 4 THP 0.970 Patient 5 TKP 2.237Patient 6 THP 0.369 Patient 7 THP 0.465

During the same analysis, 3 of the 89 control sera (blood donors) had anELISA value≧0.900 (threshold indicating a specificity of 96.6%) and nonehad an ELISA value≧1.600 (threshold indicating a specificity of 100.0%).The observed response was thus significant in patient 4 and highlysignificant in patients 1 and 5.

It is has thus been proven that it is possible to detect a significantantibody response to the polypeptides according to the invention in thecase of osteoarticular infections on prostheses which have not beendiagnosed by culturing perioperative samples.

Example B5 Protocol of the Test for Polypeptides of SEQ ID Nos. 12, 14and 16 (Western Blot)

For the Western Blot tests, sera obtained from patients having had adocumented S. aureus or S. epidermidis infection (laboratory collection)were used.

The control sera were sera obtained from blood donors (laboratorycollection) and from patients carrying other infections (laboratorycollection). Any possible binding of the antibodies present in thesesera was assessed using Western Blot tests on total extracts of bacteriaproducing polypeptides of SEQ ID Nos 12, 14 and 16.

The nitrocellulose membrane onto which the proteins of the extract weretransferred was saturated for 30 minutes using a phosphate bufferedsaline (PBS) solution containing 3% semi-skimmed milk. After threewashes with PBS containing 0.05% polyoxyethyelene sorbitan (Tween), themembrane was exposed to the test serum at the appropriate dilution ratio(i.e. 1:300) in PBS buffer containing 3% semi-skimmed milk, for 45minutes. After three new washes, goat anti-human immunoglobulin G, M orA (secondary) antibodies or, simultaneously, goat anti-humanimmunoglobulin G and/or A and/or M (secondary) antibodies labelled withalkaline phosphatase (for example 170-6462, Biorad) were added for 30minutes after having been diluted according to the supplier's protocolin PBS buffer containing 3% semi-skimmed milk. Three new washes werecarried out and then 5-bromo-4-chloro-3-indolyl phosphate and nitrobluetetrazolium were added according to the supplier's instructions untilthe result was obtained. A “positive” result corresponds toprecipitation of the substrate on the membrane at the position of thepolypeptides of SEQ ID Nos 12, 14 or 16.

The results of these tests prove the existence of a significant antibodyresponse in the case of osteoarticular infections on foreign material topolypeptides of SEQ ID Nos 12, 14 or 16 and the relevance of thesepolypeptides and of their associations/combinations for serologicaldiagnosis of this type of infection. In fact, the combination ofpolypeptides of SEQ ID Nos 12, 14 or 16 is also relevant.

Similar results were obtained for all the polypeptides according to theinvention (polypeptides of SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32 and 34).

It has thus been proven, on the one hand, that there is a significantantibody response (IgG and/or IgA) in the case of osteoarticularinfections on foreign material and, on the other hand, that thepolypeptides according to the invention and theirassociations/combinations are relevant for the serological diagnosis ofthis type of infection. In fact, the combination of at least twopolypeptides of SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32 and 34 is also relevant. Furthermore, the combination ofthe results obtained by detection of different antibody isotypes insera, for each of the polypeptides, is also relevant.

C) Use of the Polypeptides According to the Invention for TherapeuticFollow-Up Checks (Table 6)

For patients in convalescence, the observance of a decrease over time inthe ELISA values, obtained by detecting the immunoglobulins (antibodies)present in the sera by way of their binding to antigens 2B6 or 2D6-B1according to the invention, demonstrates that they are recovering well.

TABLE 6 Progression as a function of the secondary antibody used ELISAvalue 2D6B1 2B6 Patient 1 anti-IgGAM 0.99 2.3 (+) anti-IgG 0.4 0.51 (+)anti-IgA 0.66 0.34 Patient 1 + 1 year anti-IgGAM 0.44 0.74 anti-IgG 0.270.24 anti-IgA 0.31 0.31 Patient 2 anti-IgGAM 1.03 1.43 (+) anti-IgG 0.50.46 (+) anti-IgA 1.6 (+) 0.2 Patient 2 + 3 months anti-IgGAM 0.44 0.74anti-IgG 0.27 0.24 anti-IgA 0.31 0.31 Patient 3 anti-IgGAM 2.27 (+) 1.6(+) anti-IgG 0.94 (+) 0.5 (+) anti-IgA 0.14 0.19 Patient 3 + 6 monthsanti-IgGAM 2.41 (+) 0.86 anti-IgG 0.87 (+) 0.22 anti-IgA 0.15 0.19 TheELISA values marked (+) are considered to be positive

In Table 6, the individuals who were initially positive according to theinvention have become negative.

Use of the polypeptides according to the invention thus makes itpossible to carry out therapeutic follow-up checks, i.e. (i) to assessthe effect of treatment and (ii) to monitor the post-operative (orpost-implantation) progression of a patient.

D) Optimisation of Antigens and in vitro Diagnostic Tests

The study of fragments in order to improve, antigen performance on theone hand, and polypeptide production on the other is particularly usefulfor industrial use thereof and for the relevance of the tests.

Example D1 Increase in Sensitivity and Specificity

Table 7 shows the performances of 2B6, which is a fragment of E4,relative to the polypeptide E4.

TABLE 7 Optimisation of the sensitivity and specificity of E4, 2B6;ELISA results using an IgGAM secondary antibody Polypeptide tested E42B6 Percentage of “positive” sera from the 15 sera of 47.0% 67.0%patients infected with S. epidermidis diagnosed according to the priorart and who underwent the diagnostic procedure according to theinvention Percentage of “negative” sera from the 96 blood 95.0% 99.0%donor sera tested as controls

There is a substantial difference depending on the polypeptide fragmentsused. This difference was not foreseeable based on the sequences. Thesensitivity and specificity of 2B6 (fragment of E4) are thus greaterthan the entire protein or E4 (Table 7).

The use of antigen 2B6 to detect (total) immunoglobulins A, G and Mmakes it possible to identify 67% of PJIs caused by S. epidermidis (10out of the 15 test sera) with 99% specificity (1 in 96 test blood donorsera).

Example D2 Increase in Stability and Production Levels

Furthermore, protein 2B6 does not deteriorate during its production inthe host cell or during its purification.

E) Mulitparametric Combination and Evaluation by Syndrome

The combined use of a plurality of polypeptides according to theinvention also makes it possible:

-   -   (i) to increase the sensitivity of detection;    -   (ii) to assess the likelihood of the presence of a plurality of        bacteria (for example by using an antigen which is specific to a        bacteria or to a family of bacteria);    -   (iii) to identify the genotype of the strain;    -   (iv) to determine progression of the pathology (by using        specific antigens and antibody isotypes);    -   (v) to carry out therapeutic follow-up checks at reduced cost;        and    -   (vi) to carry out diagnosis per syndrome

In conclusion, the present invention discloses polypeptides havingoptimum sensitivity and specificity, enabling them to be used as a probe(antigen probe) in tests. The present invention makes it possible,without the need for surgery, to diagnose staphylococcal infections onforeign material, such as osteoarticular prostheses, in particularinfections which have not been diagnosed by culturing deep perioperatvesamples.

1-13. (canceled)
 14. A method for determining if an individual is infected by Staphylococcus aureus and/or Staphylococcus epidermidis, comprising: determining if antibodies directed against a protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, are present in a biological sample of the individual, and deducing therefrom that the individual is infected by Staphylococcus aureus and/or Staphylococcus epidermidis.
 15. The method of claim 14, wherein determining antibodies directed against a protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, are present in a biological sample of the individual comprises: contacting the biological sample with: a protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or a homologous protein comprising a sequence having at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or at least one fragment of said protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or of said homologous protein; provided said homologous protein and said at least one fragment are antigens of Staphylococcus aureus, Staphylococcus epidermidis, and/or Staphylococcus caprae; detecting antibodies bound to said protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, to said homologous protein or to said at least one fragment.
 16. The method of claim 14, wherein the biological sample is selected from the group constituted of blood, serum, urine, saliva, cerebrospinal fluid, pleural fluid, and articular fluid.
 17. A method for determining if an individual is infected by Staphylococcus aureus and/or Staphylococcus epidermidis, comprising: contacting a biological sample of the individual with: a protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or a homologous protein comprising a sequence having at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or at least one fragment of said protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or of said homologous protein; provided said homologous protein and said at least one fragment are antigens of Staphylococcus aureus, Staphylococcus epidermidis, and/or Staphylococcus caprae; detecting antibodies bound to said protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, to said homologous protein or to said at least one fragment; deducing therefrom that the individual is infected by Staphylococcus aureus and/or Staphylococcus epidermidis.
 18. The method of claim 17, wherein the biological sample is selected from the group constituted of blood, serum, urine, saliva, cerebrospinal fluid, pleural fluid, and articular fluid.
 19. A method for determining the presence of antibodies directed against Staphylococcus aureus and/or Staphylococcus epidermidis in a biological sample comprising: contacting the biological sample with: a protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or a homologous protein comprising a sequence having at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or at least one fragment of said protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or of said homologous protein; provided said homologous protein and said at least one fragment are antigens of Staphylococcus aureus, Staphylococcus epidermidis, and/or Staphylococcus caprae; detecting antibodies bound to said protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, to said homologous protein or to said at least one fragment.
 20. The method of claim 19, wherein the biological sample is selected from the group constituted of blood, serum, urine, saliva, cerebrospinal fluid, pleural fluid, and articular fluid.
 21. A pharmaceutical composition comprising: a protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or a homologous protein comprising a sequence having at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or at least one fragment of said protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or of said homologous protein; provided said homologous protein and said at least one fragment are antigens of Staphylococcus aureus, Staphylococcus epidermidis, and/or Staphylococcus caprae; as well as a pharmaceutically acceptable excipient.
 22. A method for the prevention and/or the treatment of an infection by Staphylococcus aureus and/or Staphylococcus epidermidis in an individual, comprising administering the individual with a prophylactically and/or a therapeutically effective amount of: a protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or a homologous protein comprising a sequence having at least 80% identity with a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36; or at least one fragment of said protein comprising a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or of said homologous protein; provided said homologous protein and said at least one fragment are antigens of Staphylococcus aureus, Staphylococcus epidermidis, and/or Staphylococcus caprae. 